LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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Fig. 1.4: The incomplete S phase checkpoint response. Stalled replication forks result in formation of single stranded segments during replication resulting in recruitment of Rad and Rfc family members. These activate ATM and ATR, which in turn result in activation of Chk1 and Chk3 by phosphorylation. Chk1 and Chk2 phosphorylate cdc25A, cdc25B and cdc25C resulting in generation of a 14-3-3 binding site. 14-3-3 binding results in 50
cytoplasmic accumulation of cdc25 phosphatases and also prevents interaction with their substrates, cyclin/CDK complexes. Phosphorylated cdc25A also undergoes proteolytic degradation Inhibition of cdc25 phosphatases results in inactivation of cyclinE, cyclinA and cyclin/B associated CDKs, preventing S phase and mitotic progression. 1.2.4 The G2/M DNA Damage checkpoint: The G2/M checkpoint prevents entry of the cells into mitosis when cells encounter DNA damage during the G2 phase or enter the G2 phase with damaged DNA from the previous phases of the cell cycle. The checkpoint is activated in response to DNA damaging agents such as UV, IR, adriamycin and methylmethane sulfonates (95). The main target of the G2/M checkpoint is the cyclinB/cdk1 complex whose activity is required for the entry of cells into mitosis. Checkpoint response is triggered by DNA damage which is sensed by sensor proteins, presumably belonging to the rad family (21, 92, 218, 406-408) (Figure 1.5). The signal is then communicated to transducers resulting in the activation of kinases ATM and ATR (Figure 1.5). ATM and ATR phosphorylate and activate the checkpoint kinases Chk1 and Chk2 (4, 35, 52, 111, 228, 229, 252) (Figure 1.5). Chk1 and Chk2 phosphorylate and inhibit the function of cdc25 phosphatases A, B and C (56, 120, 143, 217, 228, 241, 281, 318, 341, 370, 391) (Figure. 1.5). The inhibition of cdc25 family of phosphatases prevents dephosphorylation of cdk1 on threonine 14 and tyrosine 15, maintaining the cyclinA/cdk1 and cyclinB1/cdk1 complexes in an inactive state and preventing mitotic progression. Activated Chk1 also phosphorylates Wee1 and promotes its association with 14-3-3 proteins (203, 265) (Figure. 1.5). Phosphorylation and 14-3-3 binding seem to activate Wee1 which maintains the inhibitory phosphorylations on cdk1(203, 265) (Figure 1.5). DNA damage checkpoint response also stabilizes Wee1 levels by preventing its ubiquitin mediated proteolysis (395) (Figure 1.5). Checkpoint kinases also phosphorylate p53 resulting in stabilization of p53 and transactivation of p53 responsive genes (54, 116, 151, 232, 332, 404) (Figure.1.5). Among the genes that p53 transactivates in response to DNA damage are p21 and 14-3- 3σ (149, 216, 236, 246) (Figure 1.5). Both p21 and 14-3-3σ inhibit the activity of cyclinB1/cdk1 complex (40, 50) (Figure 1.5). p21 belongs to the Cip/Kip family CDK 51
Page 1 and 2: Generation of knockdown mice that l
Page 3 and 4: STATEMENT BY AUTHOR This dissertati
Page 5 and 6: CERTIFICATE I certify that the thes
Page 7 and 8: I am thankful to Mr Uday Dandekar f
Page 9 and 10: 3.16 Isolation of Genomic DNA (gDNA
Page 11 and 12: A synopsis of thesis to be submitte
Page 13 and 14: esults in loss of 14-3-3 binding an
Page 15 and 16: and NheI (NEB) to remove tetO-CMV,
Page 17 and 18: Hanging drop and wound healing assa
Page 19 and 20: 360). To generate transgenic animal
Page 21 and 22: asement of the seminiferous tubules
Page 23 and 24: normal female, pups were screened f
Page 25 and 26: 14-3-3ε only in the presence of HP
Page 27 and 28: proteins are expressed in testis (3
Page 29 and 30: 5. Lalit Sehgal, Srikanth B., Khyat
Page 31 and 32: Presented a poster at 33rd All Indi
Page 33 and 34: shRNA siRNA TBS-T U.V WT C IF DSG D
Page 35 and 36: List of Figures: Page No 1. Figure
Page 37 and 38: 37. Figure 4.3.20: 14-3-3γ interac
Page 39 and 40: CHAPTER 1 INTRODUCTION 39
Page 41 and 42: 1.1.1 Cyclin Cdk complexes Eukaryot
Page 43 and 44: 1.1.1.c Cyclin A cdk complexes The
Page 45 and 46: of which are associated with acquis
Page 47 and 48: 1.2.2 The G1/S DNA Damage checkpoin
Page 49: complexes. DNA damage induced degra
Page 53 and 54: 14-3-3 binding site. 14-3-3 binding
Page 55 and 56: G2 arrest and induced premature chr
Page 57 and 58: emoval of S216 phosphorylation as i
Page 59 and 60: embryogenesis, a loss of cdc25A can
Page 61 and 62: dimer was essential for Raf activat
Page 63 and 64: of enhanced apoptosis of peripheral
Page 65 and 66: the generation of transgenic mice a
Page 67 and 68: CHAPTER 2 AIMS AND OBJECTIVES 67
Page 69 and 70: CHAPTER 3 MATERIALS AND METHODS 69
Page 71 and 72: Name of oligonucleotide EF1α Forwa
Page 73 and 74: cleavage sites GAAGGTATATTGCTGTTGAC
Page 75 and 76: R E1 A GAACGAATAGTGAAGCCACAGATGTA s
Page 77 and 78: 100mm 25ug 225ul 250ul 500 1000ul T
Page 79 and 80: for the transgene were considered a
Page 81 and 82: solution in TBS-T containing 0.02%
Page 83 and 84: mounting agent. Confocal images wer
Page 85 and 86: 3.26 Antibodies used for Western bl
Page 87 and 88: each plate was added cold 500ml of
Page 89 and 90: 3.32 Hanging drop assay to determin
Page 91 and 92: 3.37 Biodistribution of 18 F -FDG u
Page 93 and 94: 2.5M NaCl 150mM 60ml Tween-20 (Sigm
Page 95 and 96: CHAPTER 4 RESULTS 95
Page 97 and 98: Figure 4.1.1 Generation of lentivir
Page 99 and 100: 4.1.2 Generation of lentiviral vect
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transduction with the virus. Untran
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Figure 4.1.6: Application of bi-cis
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Figure 4.1.7: Application of bi-cis
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Figure 4.2.1: Infection of morulae
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Figure 4.2.2: Generation of transge
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Figure 4.2.3: EGFP-f expression in
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sequenced and the distribution amon
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4.3 Generation of 14-3-3γ knockdow
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numbering). Alignment was done usin
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presence of EGFP-f by performing po
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the mouse tested infected with vect
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Figure 4.3.7: Staging of 14-3-3γ k
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containing spermatozoa is on the Y-
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Figure 4.3.9: 14-3-3γ knockdown le
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Figure 4.3.11: 14-3-3γ knockdown i
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intercellular space (12, 110, 115,
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Figure 4.3.13: 14-3-3γ loss leads
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Figure 4.3.16: Localization of PKP3
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Figure 4.3.18: Reintroduction of sh
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A B 139
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from our laboratory demonstrated th
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Figure 4.3.21: Plakoglobin recruitm
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4.3.23 (A and B)). These proteins g
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the desmosomal proteins recruitment
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Figure 4.3.25: Generation of stable
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KIF5B, however its reduction does n
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compared to vector control cells. (
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4.4 Generation of 14-3-3ε knockdow
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into the interstitial spaces of the
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Figure 4.4.3: 14-3-3ε knockdown in
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Figure 4.4.4: Patchy hair loss in 1
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follicle formation marked by black
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Figure 4.4.7: Phenotypes in 14-3-3
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compared to WT lungs (indicated by
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compared to wild type (Figure 4.4.1
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and CD4 positive lyumphocyte number
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Figure 4.4.13: Bone marrow cells fr
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Figure 4.4.15: Generation of stable
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Figure 4.4.16: NIH3T3 cells lacking
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Figure 4.4.18: Generation of induci
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CHAPTER 5 DISCUSSION 181
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to generate transgenic animals expr
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transgenic research and the use of
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(reviewed in (152, 374)). Kinesins
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subtypes known to date. Type IV col
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Figure 5.1 Schematic representation
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loss of 14-3-3ε the T cell activat
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BIBLIOGRAPHY: 1. Abraham, R. T. 200
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29. Blomer, U., L. Naldini, T. Kafr
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58. Cheng, C. Y., and D. D. Mruk. 2
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90. Elia, A. E., P. Rellos, L. F. H
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118. Girard, F., U. Strausfeld, J.
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150. Herrmann, H., M. Haner, M. Bre
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and activation of a cyclin E-cdk2 c
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213. Liu, D., J. Bienkowska, C. Pet
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242. Moll, R., W. W. Franke, D. L.
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274. Pagano, M., R. Pepperkok, F. V
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305. Russell, L. D. 1978. The blood
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inhibitor of metalloproteases-1 in
Page 219 and 220:
364. Tsunekawa, N., M. Matsumoto, S
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397. Yashiro, M., N. Nishioka, and
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Research: Biology to Prevention to
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LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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